异体CD3AK细胞治疗人肾癌SCID小鼠移植瘤模型的有效性研究

发布时间：2018-08-28 16:59

【摘要】：目的肾细胞癌(Renal cell carcinoma, RCC)占男性新发癌症的4%,在女性新发癌症病例中占3%,与其他实体肿瘤不同的是,肾癌对肿瘤的传统疗法均不敏感,如放射治疗、化学治疗等.但是肾癌被发现是免疫相关的肿瘤,多种免疫治疗在肾癌的治疗中取得了不同程度的成功。本实验通过研究过继细胞免疫治疗(Adoptive cellular immunotherapy, ACI):异体抗CD3分子的单克隆抗体激活的杀伤细胞(anti-CD3monoclonal antibody activated killer cells, CD3AK)对人肾癌细胞株OS-RC-2的体外杀伤作用,以及其对严重联合免疫缺陷(severe combined immunodeficiency, SCID)小鼠人肾癌移植瘤模型的体内抑制作用,从而进一步探讨异体CD3AK细胞对SCID小鼠人肾癌移植瘤治疗的有效性,为异体CD3AK细胞治疗肾癌的临床应用提供理论及实验支持。方法 1.人肾癌OS-RC-2细胞株的培养与传代：无菌细胞培养,及时进行换液与传代,定时观察细胞形态与数量,待细胞进入对数生长期时,台盼蓝染色鉴定活力后用于实验。 2.于体外条件下建立大量扩增异体CD3AK细胞的培养体系：采集健康志愿者的外周血,体外分离获得外周血单个核细胞(peripheral blood mononuclear cell, PBMC),培养获得异体CD3AK细胞；采用流式细胞仪检测CD3AK细胞的表型； 3.CCK-8(Cell Counting Kit-8)法检测异体CD3AK细胞对人肾癌0S-RC-2细胞株的杀伤作用； 4.建立荷瘤模型：采用左侧腹股沟皮下注射的方法,建立人肾癌SCID鼠皮下移植瘤模型,并通过尾静脉注射异体CD3AK细胞,对照组尾静脉注射磷酸盐缓冲液(phosphate buffer solution, PBS),剂量为0.2m1／只,观察小鼠的生物学特征。SCID小鼠被随机分为四组(n=8)：A组(于荷瘤前尾静脉注射异体CD3AK细胞),B组(A组对照组：荷瘤前尾静脉注射PBS)；C组(荷瘤后尾静脉注射异体CD3AK细胞),D组(C组对照组：荷瘤后尾静脉注射PBS)。隔日测量小鼠的体重、肿瘤体积以观察小鼠体重及肿瘤生长变化；末次治疗结束后24小时处死小鼠,无菌取脾脏、肿瘤组织用于后续实验。 5.免疫组化法检测CD3+细胞在小鼠肿瘤组织中的表达情况；计算脾脏指数(脾重／体重),观察异体CD3AK细胞对肾肿瘤生长的抑制作用及其对小鼠免疫系统的作用。结果 1.将异体CD3AK细胞与人肾癌OS-RC-2细胞共同孵育12h,用CCK-8法检测可以观察到,随着效应细胞与靶细胞比值的增大,异体CD3AK细胞对OS-RC-2细胞的杀伤活性随之增高。 2.A、C组小鼠与其对照组B、D组小鼠比较,A、C组小鼠的脾脏指数显著增大(P0.05)；A组与C组比较,小鼠脾脏指数无明显差异(P0.05)； 3.A、C组小鼠与其对照组B、D组小鼠比较,A、C组小鼠肿瘤重量显著减小,肿瘤生长缓慢(P0.05)；A、C组与B、D组小鼠体重均增加,实验(A、C)组小鼠体重较其对照(B、D)组小鼠体重增加的少(P0.05), 4.A组小鼠与C组小鼠比较,A组小鼠肿瘤重量减小,肿瘤生长迟缓(P0.05)。 5.免疫组化方法示,CD3+细胞在C组小鼠肿瘤组织中有阳性表达,其余各组小鼠肿瘤组织未发现有阳性表达。结论 1.异体CD3AK细胞对人肾癌OS-RC-2细胞株具有体外杀伤作用； 2.异体CD3AK细胞对人肾癌SCID小鼠移植瘤的生长具有显著抑制作用； 3.异体CD3AK细胞对人肾癌SCID小鼠移植瘤的发生具有一定的预防作用； 4.异体CD3AK细胞能增强人肾癌荷瘤SCID小鼠机体免疫功能并发挥其抗肿瘤作用。
[Abstract]:objective
Renal cell carcinoma (RCC) accounts for 4% of all new cancers in men and 3% of all new cancers in women. Unlike other solid tumors, RCC is insensitive to traditional therapies such as radiotherapy and chemotherapy. In this study, we investigated the in vitro killing effect of anti-CD3 monoclonal antibody activated killer cells (CD3AK) on human renal cell carcinoma cell line OS-RC-2 and its severe association with adoptive cell immunotherapy (ACI). The inhibitory effect of allogeneic CD3AK cells on human renal cell carcinoma xenograft tumor in SCID mice was investigated in vivo, which provided theoretical and experimental support for the clinical application of allogeneic CD3AK cells in the treatment of renal cell carcinoma.
Method
1. Culture and passage of human renal cell carcinoma OS-RC-2 cell line: aseptic cell culture, timely liquid exchange and passage, regular observation of cell morphology and quantity, when the cell entered the logarithmic growth phase, trypan blue staining identified the viability of the experiment.
2. Establish a culture system of CD3AK cells in vitro. Collect peripheral blood mononuclear cells (PBMC) from healthy volunteers and isolate PBMC in vitro to obtain allogeneic CD3AK cells. Flow cytometry was used to detect the phenotype of CD3AK cells.
3.CCK-8 (Cell Counting Kit-8) method was used to detect the killing effect of CD3AK cells on human renal cell carcinoma 0S-RC-2 cell line.
4. Establishment of tumor-bearing model: SCID mice were subcutaneously injected into the left groin to establish a model of human renal cell carcinoma. Allogeneic CD3AK cells were injected into the tail vein, and phosphate buffer solution (PBS) was injected into the tail vein of the control group. The biological characteristics of the mice were observed at a dose of 0.2m1/mouse. They were divided into four groups (n=8): group A (injecting allogeneic CD3AK cells into the anterior and caudal veins of tumor-bearing mice), group B (control group A: injecting PBS into the anterior and caudal veins of tumor-bearing mice), group C (injecting allogeneic CD3AK cells into the posterior tail veins of tumor-bearing mice) and group D (control group C: injecting PBS into the tail veins of tumor-bearing mice). The mice were sacrificed 24 hours after the end of the last treatment, and the spleen was removed by asepsis. The tumor tissues were used for subsequent experiments.
5. Immunohistochemistry was used to detect the expression of CD3+ cells in tumor tissues of mice, and spleen index (spleen weight/body weight) was calculated to observe the inhibitory effect of allogeneic CD3AK cells on the growth of renal tumor and the immune system of mice.
Result
1. Allogeneic CD3AK cells were incubated with human renal cell carcinoma OS-RC-2 cells for 12 hours. CCK-8 assay showed that the cytotoxicity of allogeneic CD3AK cells to OS-RC-2 cells increased with the increase of the ratio of effector cells to target cells.
2. The spleen index of mice in group A and C was significantly higher than that of mice in group B and D (P 0.05), and there was no significant difference between group A and group C (P 0.05).
3. Compared with the control group B, D mice, the weight of tumors in group A, C decreased significantly and the growth of tumors was slow (P 0.05); The weight of mice in group A, C and B, D increased significantly, and the weight of mice in group A and C increased less than that of mice in group B and D (P 0.05).
Compared with group C, mice in group 4.A showed less tumor weight and slower tumor growth (P0.05) than group A mice.
5. Immunohistochemical staining showed that CD3+ cells were positively expressed in tumor tissues of mice in group C, but no positive expression was found in tumor tissues of mice in other groups.
conclusion
1. allogeneic CD3AK cells have an in vitro killing effect on human renal cell carcinoma OS-RC-2 cell line.
2. allogeneic CD3AK cells significantly inhibited the growth of transplanted human renal cell carcinoma SCID mice.
3. allogeneic CD3AK cells have a certain preventive effect on the transplanted tumor of human renal carcinoma SCID mice.
4. allogeneic CD3AK cells can enhance the immune function of human renal cell carcinoma bearing SCID mice and play an anti-tumor role.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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